Bottom Line:
In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding.These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM.The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

Affiliation: Institute of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.

ABSTRACTNeural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

Figure 11: Expression of chicken axonin-1 and NgCAM in mouse DRG explants: Cultured mouse DRG explants were infected with the adenoviral vectors AdCMVax-1 (a, b, e, and f) and AdCMVNgCAM (c, d, g, and h), i–l represent uninfected controls. 60 h after infection axonin-1 and NgCAM were detected on the cell surface by indirect immunofluorescence. For immunofluorescence staining the axonin-1–specific monoclonal antibody X7C11 and the NgCAM-specific monoclonal antibody 12-I-4E-311, and a Cy3-labeled donkey anti–mouse secondary antibody were used. Axonin-1 staining: a, b, and i (fluorescence optics; e, f, and k phase optics); NgCAM staining: c, d, and j (fluorescence optics; g, h, and l phase optics). Quantitative analysis of neurite fasciculation is represented by m. In brief, for quantification individual neurites that stained positively for the heterologously expressed protein were tracked from the periphery of the ganglion to a point at one-third of the total neurite length. Neurites that coalesced into bundles consisting of more than 4 neurites were scored as the stronger fasciculated category (filled bars) and neurites found in bundles of less than 4 neurites belonged to the other category (empty bars). 50 neurites were scored per ganglion, n = 6 (6 ganglia were analyzed per sample). Bars represent means ± SD. Bars, 100 μm.

Mentions:
Overexpression of axonin-1 resulted in a slightly reduced neurite fasciculation (Fig. 11, a, b, e, and f) compared with control explants treated with AdCMVlacZ (not shown) or without virus (Fig. 11, i–m). In contrast, overexpression of NgCAM resulted in strongly enhanced fasciculation (Fig. 11, c, d, g, and h). Quantitative analysis of the effects of axonin-1 and NgCAM overexpression on neurite fasciculation are summarized in Fig. 11 m (for details see Materials and methods). The effect of NgCAM overexpression on neurite fasciculation was blocked specifically by the addition of monoclonal anti-NgCAM IgG (Fig. 12, c and f) but not by control IgG (Fig. 12, b and e). The enhanced fasciculation observed with neurites overexpressing NgCAM can therefore be ascribed specifically to the enhanced NgCAM expression in the neuronal membrane. The observed effects of axonin-1 and NgCAM overexpression on neurite fasciculation are in accordance with the observation that NgCAM but not axonin-1 can efficiently mediate contact across the extracellular space in the context of heterooligomeric axonin-1–NgCAM complexes.

Figure 11: Expression of chicken axonin-1 and NgCAM in mouse DRG explants: Cultured mouse DRG explants were infected with the adenoviral vectors AdCMVax-1 (a, b, e, and f) and AdCMVNgCAM (c, d, g, and h), i–l represent uninfected controls. 60 h after infection axonin-1 and NgCAM were detected on the cell surface by indirect immunofluorescence. For immunofluorescence staining the axonin-1–specific monoclonal antibody X7C11 and the NgCAM-specific monoclonal antibody 12-I-4E-311, and a Cy3-labeled donkey anti–mouse secondary antibody were used. Axonin-1 staining: a, b, and i (fluorescence optics; e, f, and k phase optics); NgCAM staining: c, d, and j (fluorescence optics; g, h, and l phase optics). Quantitative analysis of neurite fasciculation is represented by m. In brief, for quantification individual neurites that stained positively for the heterologously expressed protein were tracked from the periphery of the ganglion to a point at one-third of the total neurite length. Neurites that coalesced into bundles consisting of more than 4 neurites were scored as the stronger fasciculated category (filled bars) and neurites found in bundles of less than 4 neurites belonged to the other category (empty bars). 50 neurites were scored per ganglion, n = 6 (6 ganglia were analyzed per sample). Bars represent means ± SD. Bars, 100 μm.

Mentions:
Overexpression of axonin-1 resulted in a slightly reduced neurite fasciculation (Fig. 11, a, b, e, and f) compared with control explants treated with AdCMVlacZ (not shown) or without virus (Fig. 11, i–m). In contrast, overexpression of NgCAM resulted in strongly enhanced fasciculation (Fig. 11, c, d, g, and h). Quantitative analysis of the effects of axonin-1 and NgCAM overexpression on neurite fasciculation are summarized in Fig. 11 m (for details see Materials and methods). The effect of NgCAM overexpression on neurite fasciculation was blocked specifically by the addition of monoclonal anti-NgCAM IgG (Fig. 12, c and f) but not by control IgG (Fig. 12, b and e). The enhanced fasciculation observed with neurites overexpressing NgCAM can therefore be ascribed specifically to the enhanced NgCAM expression in the neuronal membrane. The observed effects of axonin-1 and NgCAM overexpression on neurite fasciculation are in accordance with the observation that NgCAM but not axonin-1 can efficiently mediate contact across the extracellular space in the context of heterooligomeric axonin-1–NgCAM complexes.

Bottom Line:
In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding.These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM.The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

Affiliation:
Institute of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.

ABSTRACTNeural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.